BIOREMEDIATION EFFECTIVENESS: REMOVING HYDROCARBONS WHILE

MINIMIZING ENVIRONMENTAL IMPACT

1
2
31
Roger C. Prince , James R. Clark and Kenneth Lee ExxonMobil Research and Engineering
Co., Annandale, NJ 08801 USA
2

ExxonMobil Research and Engineering Co., Fairfax, VA 22037, USA and

Fisheries and Oceans Canada, Bedford Institute of Oceanography, P.O. Box 1006, Dartmouth, Nova
Scotia, Canada B2Y 4A2
ABSTRACT
Since the vast majority of hydrocarbons in crude oils and refined products are biodegradable, and
hydrocarbon-degrading microbes are ubiquitous, bioremediation can be an environmentally responsible,
cost-effective way of restoring sites contaminated

with oils and fuels. Strategies aim to alleviate

constraints on microbial metabolism, either by providing electron acceptors, such as oxygen, or essential
nutrients, such as biologically available nitrogen and phosphorus. When this is done with appropriate
care, hydrocarbon biodegradation is stimulated without any additional adverse environmental impacts.
The bioremediation of oil spilled at sea that reaches shorelines typically involves the application of
fertilizers. We present the results of environmental monitoring during a major field application of this
type of bioremediation in a temperate climate, and during a smaller field trial in the Arctic. A battery of
monitoring assays showed no adverse environmental impacts of fertilizer applications, while hydrocarbon
degradation was stimulated two- to five-fold.
BIOREMEDIATION OF MARINE OIL SPILLS
Crude oils and refined products are principally hydrocarbons, and the vast majority of these are
biodegradable (1). Nevertheless, while hydrocarbons are an excellent source of carbon and energy for
the microbes that consume them, they are incomplete foods in that they do not contain significant
concentrations of other nutrients, such as nitrogen and phosphorus, which are required for microbial
growth. A significant spill usually overwhelms the natural supply of these nutrients in the environment,
and biodegradation quickly becomes nutrient limited. The bioremediation of oil spills in aerobic marine
environments has therefore focused on adding these limiting nutrients to at least partially overcome the
nutrient limitation, while at the same time minimizing any additional impact of the fertilizer addition.
After decades of laboratory research and small field trials, (reviewed in 2,3), bioremediation was first
used on a very large scale during cleanup operations following the Exxon Valdez spill in Alaska. (4,5).
More than 120 km of shoreline received bioremediation treatment (6), and a total of 48.6 tonnes of
nitrogen, in the form of urea in Inipol EAP30 (7) and ammonium nitrate in Customblen (5) was applied

over three years. Careful monitoring at three sites demonstrated that the rate of biodegradation was
stimulated between two- and five-fold.
BIOREMEDIASI TUMPAHAN MINYAK DI LAUT

Minyak mentah dan produk olahannya merupakan hidrokarbon utama, dan sebagian besar dari mereka
bersifat biodegradable ( 1 ). Namun, meskipun hidrokarbon merupakan sumber karbon dan energi untuk
mikroba melalui makanannya, hidrokarbon tidak dilengkapi dengan nutrisi lainnya, seperti nitrogen dan
fosfor , yang diperlukan untuk pertumbuhan mikroba. Sebuah tumpahan yang signifikan biasanya
menekan pasokan alami nutrisi di lingkungan, dan biodegradasi yang cepat menjadikan nutrisi terbatas.
Bioremediasi tumpahan minyak di lingkungan laut aerobik telah difokuskan pada menambahkan nutrisi
untuk di mengatasi setidaknya sebagian keterbatasan nutrisi, sementara pada saat yang sama
meminimalkan dampak tambahan dari semakin meningkatnya pupuk. Setelah dilakukan penelitian
laboratorium selama puluhan tahun serta uji coba lapangan skala kecil, (reviewed in 2,3), bioremediasi
pertama kali digunakan pada skala yang sangat besar dalam operasi pembersihan setelah tumpahan Exxon
Valdez di Alaska. (4,5). Lebih dari 120 km dari garis pantai dilakukan pemulihan dengan bioremediasi ( 6
), dan total 48,6 ton nitrogen, dalam bentuk urea di Inipol EAP30 ( 7 ) dan amonium nitrat dalam
Customblen ( 5 ) digunakan lebih dari tiga tahun. Pemantauan di tiga lokasi menunjukkan bahwa
tingkat biodegradasi dapat ditingkatkan antara dua sampai lima kali lipat.
Environmental monitoring of bioremediation of the Exxon Valdez spill.
Several papers have addressed the efficacy of careful fertilizer applications at stimulating the
biodegradation of oil in Prince William Sound (4,5,8,9). Here we focus on the monitoring efforts used to
verify that these applications did not cause any adverse environmental impact. Careful consideration of
likely possible impacts included the possibility that the fertilizer applications might be acutely toxic,
might stimulate nearshore algal blooms, or might cause the production of biosurfactants that could result
in increased removal of oil from the shorelines by tidal flushing. None of these effects was detected.

Pemantauan lingkungan bioremediasi tumpahan Exxon Valdez

Beberapa jurnal telah membahas penggunaan pupuk secara tepat

untuk meningkatkan efektifitas

biodegradasi minyak di Prince William Sound ( 4,5,8,9 ) . Di sini kita fokus pada upaya pemantauan guna
untuk membuktikan bahwa aplikasi ini tidak menyebabkan dampak lingkungan yang merugikan.
Pertimbangannya adalah apakah penggunaan pupuk mungkin beracun akut , mungkin merangsang
ganggang di dekat pantai , atau mungkin menyebabkan produksi biosurfaktan yang dapat meningkatkan
removal minyak dari garis pantai dengan pembilasan pasang surut . Tak satu pun dari efek ini terdeteksi.

Acute toxicity
The worst case of acute toxicity of fertilizer application would have been that it was all solubilized on
the first few tides, forming a highly toxic, concentrated solution which would then disperse from the
shoreline before it could significantly influence the biodegradation rates of the residual oil. Samples were
therefore collected as the tide came in, at a depth of 50 cm when the incoming tide was 1 meter deep, and
again, at the same position in the water column, as the tide withdrew. This was repeated for the second,
third, fifth and seventh outgoing tides. The water was sent to a testing laboratory for 96- hour toxicity
tests with Mysidopsis bahia (10), the most sensitive of several species tested as part of the USEPAs
initial experiments. No acute toxicity was found after fertilizer application. Furthermore, the levels of
ammonia measured in nearshore waters were in accord with the lack of toxicity. Concentrations of
ammonia peaked at up to 35M between 7 and 57 hours after fertilizer application, and then returned to
baseline levels of about 1M within four days. Such levels are below published criteria for short- term
toxicity (11).
Toksisitas Akut
Hal terburuk jika terbentuk toksisitas akut dari penggunaan pupuk akan dilarutkan pada arus pasang
pertama, membentuk larutan konsentrat yang sangat beracun yang kemudian akan menyebar dari garis
pantai sebelum toksik secara signifikan dapat mempengaruhi tingkat biodegradasi sisa minyak. Oleh
karena itu sampel diambil sebagai air pasang datang, pada kedalaman 50 cm ketika gelombang datang
dengan ketinggian 1 meter, kemudian diulangi sekali lagi di posisi yang sama di kolom air, seperti air
pasang menarik. Dan diulang lagi untuk pasang keluar kedua, ketiga, kelima dan ketujuh. Sampel air
dikirim ke laboratorium pengujian untuk uji toksisitas 96- jam dengan Mysidopsis bahia (10), yang paling
sensitif dari beberapa spesies diuji sebagai bagian dari USEPA's percobaan awal. Tidak ditemukan
toksisitas akut pada penggunaan pupuk. Selain itu, tingkat amonia diukur di perairan dekat pantai.
Konsentrasi amonia memuncak sampai dengan 35M antara 7 hingga 57 jam setelah penggunaan pupuk,
dan kemudian kembali ke tingkat dasar di sekitar 1M dalam waktu empat hari. Tingkat tersebut berada
bawah kriteria toksisitas jangka pendek (11).
Algal blooms
Just as oil-degrading microorganisms are typically limited by the availability of nitrogen and phosphorus
after an oil spill, planktonic algae are also typically nutrient limited since they have a constant source of
carbon from carbon dioxide in the air. It was therefore important to ensure that the growth of these
organisms was not significantly stimulated by the bioremediation treatment. In accord with the low levels

of ammonia reported above (12) there was no increase of chlorophyll in nearshore water, and no evidence
for a stimulation of algal productivity associated with fertilizer application.
Alga Blooms
Pertumbuhan mikroorganisme pengurai minyak biasanya dibatasi oleh ketersediaan nitrogen dan fosfor
setelah tumpahan minyak , nutrisi alga planktonik biasanya juga terbatas karena mereka memiliki sumber
karbon tetap dari karbon dioksida di udara. Oleh karena itu penting untuk memastikan bahwa
pertumbuhan alga tidak didorong secara signifikan akibat bioremediasi. Rendahnya tingkat amonia sesuai
pembahasan di atas (12), serta tidak ada peningkatan klorofil di perairan dekat pantai, membuktikan tidak
adanya stimulasi produktivitas alga yang terkait dengan aplikasi pupuk.
Biosolubilization
The possibility that increased microbial activity might stimulate oil release into nearshore waters was
assessed by infrared spectroscopy. Only 16 of 174 samples collected from the three monitoring sites had
detectable levels of oil (>0.2 mg total petroleum hydrocarbons per liter), and none was higher than 0.41
mg/l. Furthermore, samples with detectable oil were not correlated with fertilized or unfertilized portions
of the shorelines. Thus no significant release of oil associated with fertilizer application was observed.
Kemungkinan bahwa peningkatan aktivitas mikroba mungkin merangsang pelepasan minyak ke perairan
dekat pantai dinilai dengan spektroskopi inframerah . Hanya 16 dari 174 sampel yang dikumpulkan dari
tiga lokasi pemantauan memiliki tingkat terdeteksi minyak ( > 0,2 mg Total hidrokarbon minyak bumi per
liter ) , dan tidak ada yang lebih tinggi dari 0,41 mg / l . Selanjutnya , sampel dengan minyak terdeteksi
tidak berkorelasi dengan bagian pupuk atau yang tidak mengandung pupuk dari garis pantai . Dengan
demikian tidak ada hasil yang signifikan dari minyak terkait dengan penggunaan pupuk yang diamati.
Environmental monitoring of bioremediation of an experimental oilspill on Spitsbergen.
An international consortium carried out a field trial of bioremediation on an Arctic beach on Spitsbergen
(78N, 17E), using an intermediate fuel oil and soluble and slow release fertilizers (13). While the
primary mechanism of oil removal from the shoreline was physical, in association with fine sediment
particles (14), there was good evidence that biodegradation was stimulated approximately two-fold for
more than a year by fertilizer applications in the first month after the spill (15). Again, here we focus on
efforts to ensure that the fertilizer applications did not cause any adverse environmental impacts. We
focused on measuring whether bioremediation was accompanied by any increased toxicity in the
sediments, or increased bioavailability of oil components to nearshore biota.

Pemantauan

lingkungan

dari bioremediasi tumpahan

minyak

eksperimental di

Spitsbergen .
Sebuah konsorsium internasional melakukan uji coba lapangan bioremediasi di pantai Arktik di
Spitsbergen ( 78 N , 17'E ) , menggunakan bahan bakar minyak kelas menengah dan larut dan lambat
melepaskan pupuk (13) . Sementara mekanisme utama dari penghapusan minyak dari garis pantai adalah
berupa fisik , berkaitan dengan partikel sedimen halus (14), ada bukti kuat bahwa biodegradasi
ditingkatkan sekitar dua kali lipat selama lebih dari satu tahun dengan penggunaan pupuk pada bulan
pertama setelah terjadi tumpahan (15) . Sekali lagi, di sini kita fokus pada upaya untuk memastikan
bahwa penggunaan pupuk tidak menyebabkan dampak lingkungan yang merugikan. Kami fokus pada
pengukuran apakah bioremediasi disertai oleh peningkatan toksisitas dalam sedimen, atau peningkatan
bioavailabilitas komponen minyak untuk biota perairan dekat pantai.
Sediment toxicity
Potential toxicity of beach sediments was monitored with the Microtox

solid phase test (16, 17) that is

a modification of that used by True and Heyward (18). Essentially the sediment is exhaustively extracted
with methylene chloride, the methylene chloride is replaced by ethanol, and dilutions are made with
saline so that the final Microtox assay contains <1% ethanol. The bioassay is based on the measurement
of changes in light emission by a non-pathogenic, bioluminescent marine bacterium (Vibrio fisheri) upon
exposure to the test samples. The Microtox

Test has been used extensively worldwide over the last 18

years for toxicity screening of chemicals, effluents, water and sediment, and for contamination surveys
and environmental risk assessment. Its application for monitoring the efficacy of oil spill remediation
methods has been proven. Our focus was on any increased environmental impact due to the application of
fertilizers, so our samples were all collected from oiled parts of the experimental plots. Acute sediment
toxicity was observed in all of the oiled experimental plots. While toxicity was highest on the fertilized
portion of the beach on Days 0 and 1 (seven days after oil was applied to the shoreline), by Day 5 it had
dropped substantially so that there was no difference in the Microtox response of samples from the
fertilized or the unfertilized plots. We attribute the initial toxic response to the elevated oil concentration
rather than to an effect due to the generation of toxic metabolites by the bioremediation strategy, because
it is unlikely that such compounds would accumulate and disappear so rapidly in this Arctic environment.
We note that while the Microtox

Test was not selected to assess the toxicity of the inorganic fertilizer

components themselves, previous studies in Prince William Sound suggest that toxic effects resulting from
the fertilizer are unlikely.

Toksisitas endapan
Potensi toksisitas sedimen pantai dipantau dengan tes Microtox

fase padat (16, 17) yang merupakan

modifikasi dari yang digunakan oleh True dan Heyward (18). Pada dasarnya sedimen diekstraksi dengan
metilen klorida, metilen klorida digantikan oleh etanol dan diencerkan dengan garam sehingga akhir uji
MICROTOX mengandung <1% etanol. Bioassay berdasarkan pada pengukuran perubahan emisi cahaya
oleh non-patogenik, bakteri laut bioluminescent (Vibrio fisheri) setelah terpapar sampel uji. Uji
Microtox

telah digunakan secara luas di seluruh dunia selama 18 tahun terakhir untuk menyaring

toksisitas bahan kimia, limbah, air dan sedimen, dan untuk survei kontaminasi dan penilaian risiko
lingkungan. Penggunaan untuk memantau efektivitas metode remediasi tumpahan minyak telah terbukti.
Fokus kami adalah pada setiap peningkatan dampak lingkungan akibat penggunaan pupuk, sehingga
sampel kami semua dikumpulkan dari subjek percobaan yang terpapar minyak. Toksisitas sedimen akut
diamati pada semua subjek percobaan yang terpapar minyak. Sementara toksisitas tertinggi pada bagian
pupuk dari pantai di hari ke-0 dan 1 (tujuh hari setelah tumpahan minyak di garis pantai) hingga hari ke-5.
Hal tersebut telah menurunkan substansi sehingga tidak ada perbedaan dalam respon MICROTOX
sampel dari pupuk atau plot yang tidak mengandung unsure pupuk. Kami tandai respon awal racun untuk
konsentrasi minyak yang ditinggikan lebih baik dari efek wajar untuk metabolit generasi beracun oleh
strategi bioremediasi, karena tidak mungkin jika senyawa tersebut akan terakumulasi dan menghilang
begitu cepat di lingkungan Kutub Utara ini. Kami mencatat bahwa sementara Uji Microtox

tidak dipilih

untuk menentukan nilai toksisitas komponen pupuk anorganik sendiri, penelitian sebelumnya di Prince
William Sound menunjukkan bahwa efek toksik yang dihasilkan dari pupuk tidak mungkin
Biosolubilization
To assess changes in the bioavailability and toxicity of residual oil components leaving the beach
sediments following treatment, we deployed semi-permeable membrane devices (SPMDs) on the beaches.
These passive in situ samplers (19) contain purified triolein (1 ml), a substance that constitutes a major
fraction of the neutral lipid of fish. When immersed in water, SPMDs absorb nonionic, organic chemicals
having a log Kow>1, a size <1 and a molecular weight of about 600 or less, as well as possible neutral
organo-metal complexes. These characteristics correspond to those of known mixed-function oxygenaseinducing compounds, including polyaromatic hydrocarbons. The operational advantage of SPMDs is that
they can provide an integrated sample over time, making them highly advantageous for field use, such as
on Spitsbergen, where site access is limited by logistical constraints. In terms of ecological relevance, the
diffusion of dissolved neutral organic chemicals into the triolein through the pores of the polyethylene
membrane within SPMDs simulates the diffusion of compounds across a live fish gill membrane. For the
Spitsbergen experiment, SPMDs were used as concentrating devices for biological testing with the

Microtox

Assay. After exposure on the shoreline the sampling tubes were frozen until analysis, then

washed sequentially with water, methylene chloride, acetone and isopropanol before being air-dried. The
lipid content was extracted with a 1:1 acetone dimethylsulfoxide mixture, which was then evaporated,
and the acute toxicity of the extracts was determined using the Microtox assay. The semi-permeable
membrane devices did not reveal any increase in toxicity over that experienced at an unoiled, although
not necessarily pristine, site within the study area
Biosolubilization
Untuk menilai perubahan bioavailabilitas dan toksisitas komponen sisa minyak yang meninggalkan
sedimen pantai setelah perawatan, kami mengerahkan perangkat membran semipermeabel (SPMDs) di
pantai. Ini pasif dalam sampler situ (19) berisi triolein murni (1 ml), zat yang merupakan fraksi utama dari
lipid netral ikan. Ketika direndam dalam air, SPMDs menyerap nonionik, bahan kimia organik memiliki
log Kow> 1, ukuran <1 dan berat molekul sekitar 600 atau kurang, serta mungkin kompleks organologam netral. Karakteristik ini sesuai dengan orang-orang dari campuran-fungsi oxygenase-merangsang
senyawa yang dikenal, termasuk hidrokarbon polyaromatic. Keuntungan operasional SPMDs adalah
bahwa mereka dapat memberikan sampel terintegrasi dari waktu ke waktu, membuat mereka sangat
menguntungkan untuk digunakan lapangan, seperti di Spitsbergen, di mana akses situs dibatasi oleh
kendala logistik. Dalam hal relevansi ekologi, penyebaran bahan kimia organik netral terlarut ke dalam
triolein melalui pori-pori membran polyethylene dalam SPMDs mensimulasikan penyebaran senyawa
melintasi membran insang ikan hidup. Untuk percobaan Spitsbergen, SPMDs digunakan sebagai
konsentrat perangkat untuk pengujian biologis dengan Microtox

Assay. Setelah paparan di garis pantai

tabung sampel dibekukan sampai dianalisa, kemudian dicuci secara berurutan dengan air, metilen klorida,
aseton dan isopropanol sebelum mongering di udara. Kandungan lipid diekstraksi dengan 1: 1 aseton
campuran dimetilsulfoksida, yang kemudian menguap, dan ekstrak toksisitas akut ditentukan
menggunakan uji MICROTOX. Perangkat membran semi-permeabel tidak memperlihatkan peningkatan
toksisitas lebih dari hasil uji pada objek yang tidak terpapar minyak, meskipun tidak selalu murni, area
masih dalam wilayah studi.
DISCUSSION
Bioremediation of oil in and on aerobic shorelines has been demonstrated to be an effective and
environmentally benign technology (2-9), but questions remain as to whether stimulating the
biodegradation of oil on shorelines has transiently adverse effects. There is ample evidence that
bioremediation removes hydrocarbons, and their associated toxicity, in both terrestrial and marine
environments (20, 21), but there are concerns that some laboratory experiments have measured transient
increases in toxicity in the early stages of biodegradation (22,23). It is important to recognize that such

toxicity seems to be associated with metabolites of the hydrocarbons (24,25), and that these are
themselves biodegradable. Thus their accumulation in the laboratory experiments reflects a transient
imbalance between different trophic guilds in the biodegradative community. Such imbalances need not
occur in the natural environment, where the rates of biodegradation are typically slower than in the
laboratory, and where indigenous microbes are likely to be much more diverse than those in captivity.
Our monitoring of bioremediation strategies during the large-scale operation in Alaska and the field
trial on Spitsbergen did not detect any transient increases of toxicity after the initiation
bioremediation.

of

While it is important to continue this research as a precautionary approach, it is

also important not to overemphasize potential problems in light of the demonstrated success of the
technology. It is also important to bear in mind that biodegradation occurs in the absence of any
bioremediation treatment. Based on actual case studies to date, no detrimental treatment effects have
been observed with the application of bioremediation strategies. Detrimental effects from the addition of
bioremediation agents have only been demonstrated in the field during controlled experiments that
assessed an unconventional bioremediation product which altered the physical/chemical properties of the
residual oil (26); or knowingly utilized excessive concentrations of organic nutrients (17).
Bioremediasi minyak pada garis pantai aerobik telah dibuktikan untuk menjadi teknologi yang efektif dan
ramah lingkungan (2-9), tapi pertanyaannya apakah meningkatkan efektifitas biodegradasi minyak di
garis pantai memiliki dampak tambahan yang merugikan. Ada banyak bukti bahwa bioremediasi
menghilangkan hidrokarbon, dan toksisitas yang terkait, baik dalam lingkungan darat dan laut (20, 21),
tetapi ada kekhawatiran bahwa beberapa percobaan laboratorium telah mengukur sementara peningkatan
toksisitas pada tahap awal biodegradasi (22,23 ). Hal ini penting untuk mengetahui bahwa toksisitas
tersebut tampaknya terkait dengan metabolit dari hidrokarbon (24,25), dan hidrokarbon sendiri bersifat
biodegradable. Sehingga

ketika terakumulasi dalam percobaan laboratorium

menggambarkan

ketidakseimbangan sementara antara serikat trofik yang berbeda di kelompok biodegradatif.

Ketidakseimbangan tersebut tidak diharapkan terjadi dalam lingkungan alam, di mana tingkat
biodegradasi biasanya lebih lambat daripada di laboratorium, dan di mana mikroba adat cenderung jauh
lebih beragam daripada yang "di penangkaran". Pemantauan kami dari strategi bioremediasi selama
operasi besar-besaran di Alaska dan uji coba lapangan di Spitsbergen tidak mendeteksi kenaikan
sementara toksisitas setelah terjadi bioremediasi. Selain untuk melanjutkan penelitian ini sebagai
pendekatan pencegahan, penelitian ini juga penting untuk tidak terlalu menekankan potensi masalah
dalam membuktikan keberhasilan teknologi. Hal ini juga penting untuk diingat bahwa biodegradasi
terjadi pada tidak adanya perawatan bioremediasi. Berdasarkan studi kasus aktual sampai saat ini, tidak
ada dampak merugikan dengan penerapan strategi bioremediasi. Dampak merugikan dari penambahan
agen bioremediasi hanya ditunjukkan di lapangan selama percobaan terpantau yang dihasilkan produk

bioremediasi konvensional yang mengubah sifat fisik / kimia dari minyak sisa (26), atau sengaja
digunakan konsentrasi berlebihan dari nutrisi organik (17).
Oil spill bioremediation epitomizes modern environmental technologies; working with natural processes
to remove spilled oil from the environment while minimizing undesirable environmental impacts. Careful
application of fertilizers can stimulate oil biodegradation two- to five-fold, with no adverse
environmental impacts. Though this stimulation may seem rather small, it can translate into oil being
removed months

to

years earlier than it would otherwise have been removed. Furthermore,

bioremediation may make a significant contribution towards accelerated habitat recovery by the removal
of toxic hydrocarbon components to levels below the toxicity threshold. Bioremediation is thus an
important part of the toolkit available for dealing with accidental and deliberate oil releases into the
marine environment.
Bioremediasi tumpahan minyak melambangkan teknologi lingkungan modern, bekerja dengan proses
alami untuk menghilangkan minyak tumpah dari lingkungan sambil meminimalkan dampak lingkungan
yang tidak diinginkan. Penggunaan pupuk secara hati-hati dapat meningkatkan biodegradasi minyak
hingga dua sampai lima kali lipat , dengan tidak ada dampak lingkungan yang merugikan. Meskipun
stimulasi ini mungkin tampak agak kecil, hal itu dapat diartikan minyak yang diuraikan bulan ke tahun
lebih awal dari itu akan dinyatakan telah dihapus. Selanjutnya, bioremediasi dapat berkontribusi secara
signifikan dengan mempercepat pemulihan habitat dengan penghapusan komponen hidrokarbon beracun
ke tingkat bawah ambang batas toksisitas. Dengan demikian bioremediasi merupakan bagian penting dari
sarana yang tersedia untuk mengurusi kejadian tumpahan minyak yang disengaja maupun tidak disengaja
ke dalam lingkungan laut.
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3. Lee, K. and deMora, S., In situ bioremediation strategies for oiled shoreline environments. Environ.
Technol. 20, . 783-794 (1999).
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Exxon Valdez oil spill. Nature 368, 413-418 (1994).
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Peltier, W.H. and Weber, C.I., Methods for measuring the acute toxicity of effluents to

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